Akt is an endogenous inhibitor toward tumor necrosis factor-related apoptosis inducing ligand-mediated apoptosis in rheumatoid synovial cells

Akt is known to be activated in the rheumatoid synovial tissues. We examined here functional role of Akt during tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in rheumatoid synovial cells. Rheumatoid synovial cells in vitro were rapidly committed to apoptosis in response to TRAIL in mitochondria-dependent manner whereas Akt and extracellular signal-regulated kinase (ERK) were also phosphorylated. TRAIL-mediated apoptosis in synovial cells was significantly increased through inactivation of Akt by LY294002, however, that process was not so changed by adding ERK inhibitor, PD98059. Platelet-derived growth factor (PDGF) clearly phosphorylated both Akt and ERK in synovial cells, and PDGF pretreatment markedly suppressed TRAIL-mediated synovial cell apoptosis. The use of not PD98059 but LY294002 abrogated PDGF-mediated inhibitory effect toward TRAIL-induced apoptosis in synovial cells. The above protective effect of Akt was confirmed by the use of short interfering RNA (siRNA)-directed inhibition of Akt. Our data suggest that Akt is an endogenous inhibitor during TRAIL-mediated synovial cell apoptotic pathway, which may explain that synovial cells in situ of the rheumatoid synovial tissues are resistant toward apoptotic cell death in spite of death receptor expression.     

Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway.

Upon detachment from the extracellular matrix, epithelial cells enter into programmed cell death, a phenomenon known as anoikis, ensuring that they are unable to survive in an inappropriate location. Activated ras oncogenes protect cells from this form of apoptosis. The nature of the survival signals activated by integrin engagement and usurped by oncogenic Ras are unknown: here we show that in both cases phosphoinositide 3-OH kinase (PI 3-kinase), but not Raf, mediates this protection, acting through protein kinase B/Akt (PKB/Akt). Constitutively activated PI 3-kinase or PKB/Akt block anoikis, while inhibition of PI 3-kinase abrogates protection by Ras, but not PKB/Akt. Inhibition of either PI 3-kinase or PKB/Akt induces apoptosis in adherent epithelial cells. Attachment of cells to matrix leads to rapid elevation of the levels of PI 3-kinase lipid products and PKB/Akt activity, both of which remain high in Ras-transformed cells even in suspension. PI 3-kinase acting through PKB/Akt is therefore implicated as a key mediator of the aberrant survival of Ras-transformed epithelial cells in the absence of attachment, and mediates matrix-induced survival of normal epithelial cells.     

Analysis of Ras-dependent signals that prevent caspase-3 activation and apoptosis induced by cytokine deprivation in hematopoietic cells

In hematopoietic cells, Ras has been implicated in signaling pathways that prevent apoptosis triggered by deprivation of cytokines, such as interleukin-3 (IL-3). However, the mechanism whereby Ras suppresses cell death remains incompletely understood. We have investigated the role of Ras in IL-3 signal transduction by using the cytokine-dependent BaF3 cell line. Herein, we show that the activation of the pro-apoptotic protease caspase-3 upon IL-3 removal is suppressed by expression of activated Ras, which eventually prevents cell death. For caspase-3 suppression, the Raf/extracellular signal-regulated kinase (ERK)- or phosphatidylinositol 3-kinase (PI3-K)/Akt-mediated signaling pathway downstream of Ras was required. However, inhibition of both pathways did not block activated Ras-dependent suppression of cell death-associated phenotypes, such as nuclear DNA fragmentation. Thus, a pathway that is independent of both Raf/ERK and PI3-K/Akt pathways may function downstream of Ras, preventing activated caspase-3-initiated apoptotic processes. Conditional activation of c-Raf-1 also suppressed caspase-3 activation and subsequent cell death without affecting Akt activity, providing further evidence for a PI3-K/Akt-independent mechanism.     

Molecular mechanisms contributing to glutamine-mediated intestinal cell survival

Glutamine, the most abundant amino acid in the bloodstream, is the preferred fuel source for enterocytes and plays a vital role in the maintenance of mucosal growth. The molecular mechanisms regulating the effects of glutamine on intestinal cell growth and survival are poorly understood. Here, we show that addition of glutamine (1 mmol/l) enhanced rat intestinal epithelial (RIE)-1 cell growth; conversely, glutamine deprivation increased apoptosis as noted by increased DNA fragmentation and caspase-3 activity. To delineate signaling pathways involved in the effects of glutamine on intestinal cells, we assessed activation of extracellular signal-related kinase (ERK), protein kinase D (PKD), and phosphatidylinositol 3-kinase (PI3K)/Akt, which are important pathways in cell growth and survival. Addition of glutamine activated ERK and PKD in RIE-1 cells after a period of glutamine starvation; inhibition of ERK, but not PKD, increased cell apoptosis. Conversely, glutamine starvation alone increased phosphorylated Akt; inhibition of Akt enhanced RIE-1 cell DNA fragmentation. The role of ERK was further delineated using RIE-1 cells stably transfected with an inducible Ras. Apoptosis was significantly increased following ERK inhibition, despite Ras activation. Taken together, these results identify a critical role for the ERK signaling pathways in glutamine-mediated intestinal homeostasis. Furthermore, activation of PI3K/Akt during periods of glutamine deprivation likely occurs as a protective mechanism to limit apoptosis associated with cellular stress. Importantly, our findings provide novel mechanistic insights into the antiapoptotic effects of glutamine in the intestine.     

The Ras/phosphatidylinositol 3-kinase and Ras/ERK pathways function as independent survival modules each of which inhibits a distinct apoptotic signaling pathway in sympathetic neurons

Ras promotes robust survival of many cell systems by activating the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, but little is understood about the survival functions of the Ras/ERK pathway. We have used three different effector-loop mutant forms of Ras, each of which activates a single downstream effector pathway, to dissect their individual contributions to survival of nerve growth factor (NGF)-dependent sympathetic neurons. The PI3-kinase pathway-selective protein Ras(Val-12)Y40C was as powerful as oncogenic Ras(Val-12) in preventing apoptosis induced by NGF deprivation but conferred no protection against apoptosis induced by cytosine arabinoside. Identical results were obtained with transfected Akt. In contrast, the ERK pathway-selective protein Ras(Val-12)T35S had no protective effects on NGF-deprived neurons but was almost as strongly protective as Ras(Val-12) against cytosine arabinoside-induced apoptosis. The protective effects of Ras(Val-12)T35S against cytosine arabinoside were completely abolished by the ERK pathway inhibitor PD98059. Ras(Val-12)E37G, an activator of RalGDS, had no survival effect on either death pathway, similar to RasS17N, the full survival antagonist. Thus, Ras provides two independent survival pathways each of which inhibits a distinct apoptotic mechanism. Our study presents one of the few clear-cut cases where only the Ras/ERK, but not the Ras/PI3K/Akt pathway, plays a dominant survival signaling role.     

Suppression of Ras-Induced Apoptosis by the Rac GTPase

Ras is an essential component of signal transduction pathways that control cell proliferation, differentiation, and survival. In this study we have examined the cellular responses to high-intensity Ras signaling. Expression of increasing amounts of the oncogenic form of human HRas, HRasV12, results in a dose-dependent induction of apoptosis in both primary and immortalized cells. The induction of apoptosis by HRasV12 is blocked by activated Rac and potentiated by dominant interfering Rac. The ability of Rac to suppress Ras-induced apoptosis is dependent on effector pathway(s) controlled by the insert region and is linked to the activation of NF-kappaB. The apoptotic effect of HRasV12 requires the activation of both the ERK and JNK mitogen-activated protein kinase cascade and is independent of p53. These results demonstrate a role for Rac in controlling signals that are necessary for cell survival, and suggest a mechanism by which Rac activity can confer growth advantage to cells transformed by the ras oncogene.     

Conditional apoptosis induced by oncogenic ras in thyroid cells

Mutations of ras are tumor-initiating events for many cell types, including thyrocytes. To explore early consequences after oncogenic Ras activation, we developed a doxycycline-inducible expression system in rat thyroid PCCL3 cells. Beginning 3-4 days after H-Ras(v12) expression, cells underwent apoptosis. The H-Ras(v12) effects on apoptosis were decreased by a mitogen-activated protein kinase kinase (MEK1) inhibitor and recapitulated by doxycycline-inducible expression of an activated MEK1 mutant (MEK1(S217E/S221E)). As reported elsewhere, acute expression of H-Ras(v12) also induces mitotic defects in PCCL3 cells through ERK (extracellular ligand-regulated kinase) activation, suggesting that apoptosis may be secondary to DNA damage. However, acute activation of SAPK/JNK (stress-activated protein kinase/Jun N-terminal kinase) through acute expression of Rac1(v12) also triggered apoptosis, without inducing large-scale genomic abnormalities. H-Ras(v12)-induced apoptosis was dependent on concomitant activation of cAMP by either TSH or forskolin, in a protein kinase A-independent manner. Thus, coactivation of cAMP-dependent pathways and ERK or JNK (either through H-Ras(v12), Rac1(v12), or MEK1(S217E/S221E)) is inconsistent with cell survival. The fate of thyrocytes within the first cell cycles after expression of oncogenic Ras is dependent on ambient TSH levels. If both cAMP and Ras signaling are simultaneously activated, most cells will die. Those that survive will eventually lose TSH responsiveness and/or inactivate the apoptotic cascade through secondary events, thus enabling clonal expansion.     

Oncogenic Ras inhibits Fas ligand-mediated apoptosis by downregulating the expression of Fas

Tumor growth is the result of deregulated tissue homeostasis which is maintained through the delicate balance of cell growth and apoptosis. One of the most efficient inducers of apoptosis is the death receptor Fas. We report here that oncogenic Ras (H-Ras) downregulates Fas expression and renders cells of fibroblastic and epitheloid origin resistant to Fas ligand-induced apoptosis. In Ras-transformed cells, Fas mRNA is absent. Inhibition of DNA methylation restores Fas expression. H-Ras signals via the PI 3-kinase pathway to downregulate Fas, suggesting that the known anti-apoptotic effect of the downstream PKB/Akt kinase may be mediated, at least in part, by the repression of Fas expression. Thus, the oncogenic potential of H-ras may reside on its capacity not only to promote cellular proliferation, but also to simultaneously inhibit Fas-triggered apoptosis.     

Oncogene toxicity in thyroid carcinomas and other types of tumors

Most studies evidenced that the Ras/Raf/MEK/ERK signaling pathway promotes proliferation and malignant transformation as a result of stimulation of cell growth and division and apoptosis prevention. The PI3K/PDK/Akt signaling pathway is involved in regulation of protein synthesis and supplying cells with energy. In addition, this pathway inhibits apoptosis, promotes cancer cell survival, and is activated in many types of tumors. However, hyperactivity of these pathways in tumor tissues can lead to oncogene induced senescence(OIS), growth arrest, and apoptosis. This phenomenon was named Toxicity of Oncogenes. The review discusses the meaning and mechanisms of oncogenic toxicity in thyroid tumors and other types of cancer.     

Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance

Growth factors and mitogens use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their receptors to regulate gene expression and prevent apoptosis. Some components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf). Mutations also occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. Even in the absence of obvious genetic mutations, this pathway has been reported to be activated in over 50% of acute myelogenous leukemia and acute lymphocytic leukemia and is also frequently activated in other cancer types (e.g., breast and prostate cancers). Importantly, this increased expression is associated with a poor prognosis. The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of activated Akt to phosphorylate and inactivate different Rafs. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell lineage specific effects. For example, Raf/MEK/ERK is usually associated with proliferation and drug resistance of hematopoietic cells, while activation of the Raf/MEK/ERK cascade is suppressed in some prostate cancer cell lines which have mutations at PTEN and express high levels of activated Akt. Furthermore the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways also interact with the p53 pathway. Some of these interactions can result in controlling the activity and subcellular localization of Bim, Bak, Bax, Puma and Noxa. Raf/MEK/ERK may promote cell cycle arrest in prostate cells and this may be regulated by p53 as restoration of wild-type p53 in p53 deficient prostate cancer cells results in their enhanced sensitivity to chemotherapeutic drugs and increased expression of Raf/MEK/ERK pathway. Thus in advanced prostate cancer, it may be advantageous to induce Raf/MEK/ERK expression to promote cell cycle arrest, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK induced proliferation and drug resistance. Thus the Raf/MEK/ERK pathway has different effects on growth, prevention of apoptosis, cell cycle arrest and induction of drug resistance in cells of various lineages which may be due to the presence of functional p53 and PTEN and the expression of lineage specific factors.     

Signal transduction of MEK/ERK and PI3K/Akt activation by hypoxia/reoxygenation in renal epithelial cells

The extracellular signal-regulated kinase (ERK) and Akt have been reported to be activated by ischemia/reperfusion in vivo. However, the signaling pathways involved in activation of these kinases and their potential roles were not fully understood in the postischemic kidney. In the present study, we observed that these kinases are activated by hypoxia/reoxygenation (H/R), an in vitro model of ischemia/reperfusion, in opossum kidney (OK) cells and elucidated the signaling pathways of these kinases. ERK and Akt were transiently activated during the early phase of reoxygenation following 4-12h of hypoxia. The ERK activation was inhibited by U0126, a specific inhibitor of ERK upstream MAPK/ERK kinase (MEK), but not by LY294002, a specific inhibitor of phosphoinositide 3-kinase (PI3K), whereas Akt activation was blocked by LY294002, but not by U0126. Inhibitors of epidermal growth factor receptor (EGFR) (AG 1478), Ras and Raf, as well as antioxidants inhibited activation of ERK and Akt, while the Src inhibitor PP2 had no effect. PI3K/Akt activation was shown to be associated with up-regulation of X chromosome-linked inhibitor of apoptosis (XIAP), but not survivin. Reoxygenation following 4-h hypoxia-stimulated cell proliferation, which was dependent on ERK and Akt activation and was also inhibited by antioxidants and AG 1478. Taken together, these results suggest that H/R induces activation of MEK/ERK and PI3K/Akt/XIAP survival signaling pathways through the reactive oxygen species-dependent EGFR/Ras/Raf cascade. Activation of these kinases may be involved in the repair process during ischemia/reperfusion.     

Activation of Ras/Raf protects cells from melanoma differentiation-associated gene-5-induced apoptosis

Melanoma differentiation-associated gene-5 (mda-5) was the first molecule identified in nature whose encoded protein embodied the unique structural combination of an N-terminal caspase recruitment domain and a C-terminal DExD/H RNA helicase domain. As suggested by its structure, cumulative evidences documented that ectopic expression of mda-5 leads to growth inhibition and/or apoptosis in various cell lines. However, the signaling pathways involved in mda-5-mediated killing have not been elucidated. In this study, we utilized either genetically modified cloned rat embryo fibroblast cells overexpressing different functionally and structurally distinct oncogenes or human pancreatic and colorectal carcinoma cells containing mutant active ras to resolve the role of the Ras/Raf signaling pathway in mda-5-mediated growth inhibition/apoptosis induction. Rodent and human tumor cells containing constitutively activated Raf/Raf/MEK/ERK pathways were resistant to mda-5-induced killing and this protection was antagonized by intervening in this signal transduction cascade either by directly inhibiting ras activity using an antisense strategy or by targeting ras-downstream factors, such as MEK1/2, with the pharmacological inhibitor PD98059. The present findings provide a further example of potential cross-talk between growth-inhibitory and growth-promoting pathways in which the ultimate balance of these factors defines cellular homeostasis, leading to survival or induction of programmed cell death.     

Identification of a novel ligand-receptor pair constitutively activated by ras oncogenes

The Ras signaling pathway is thought to control the expression of a subset of yet to be defined genes that are crucial for cell growth and differentiation. Here we have identified by differential display a novel oncogenic Ras target, mob-5, encoding a 23-kDa cytokine-like secreted protein. Mob-5 expression could be induced by oncogenic Ha-ras and Ki-ras, but not by normal ras activation. Inhibitors of both Ha-Ras and mitogen-activated protein kinase kinase completely abolished the mob-5 expression in ras transformed cells, with concomitant loss of the transformation phenotype. Using an alkaline phosphatase-tagged Mob-5 as ligand, a putative Mob-5 receptor was identified on the cell surface of oncogenic ras transformed cells. Thus, the Mob-5/Mob-5 receptor may represent a novel putative autocrine loop coordinately activated by ras oncogenes.     

Activation of extracellular signal-regulated kinase, ERK2, by p21ras oncoprotein.

To examine signal transduction events activated by oncogenic p21ras, we have studied kinases that are activated following the scrape loading of p21ras into quiescent cells. We observe rapid activation of 42 kDa and 46 kDa protein kinases. The 42 kDa kinase is the mitogen and extracellular-signal regulated kinase ERK2, (MAP2 kinase), which is activated by phosphorylation on tyrosine and threonine in response to oncogenic p21ras, while the 46 kDa kinase is likely to be another member of the ERK family. Stimulation of these kinases by oncogenic p21ras does not require the presence of growth factors, showing that oncogenic p21ras uncouples kinase activation from external signals. In ras transformed cell lines, these kinases are constitutively activated. We propose that the kinases are important components of the signal transduction pathway activated by p21ras oncoprotein.     

Synergy between PI3K/Akt and Raf/MEK/ERK pathways in IGF-1R mediated cell cycle progression and prevention of apoptosis in hematopoietic cells

The insulin like growth factor-1 (IGF-1) receptor (R) induced PI3K/Akt signal transduction cascade has critical roles in prevention of apoptosis and regulation of cell cycle progression. Here, we discuss the effects of IGF-1R-mediated signal transduction on hematopoietic cells which normally require interleukin-3 (IL-3) for growth and prevention of apoptosis. Cytokine-dependent FDC-P1 hematopoietic cells were conditionally transformed to grow in response to overexpression of IGF-1R in the presence of IGF-1. When these cells were deprived of IL-3 or IGF-1 for 24 hrs, they exited the cell cycle, activated caspase 3 and underwent apoptosis. The effects of inhibitors which targeted the PI3K/Akt and Raf/MEK/ERK pathways were determined. When the cells were cultured with IGF-1 and either PI3K or MEK inhibitors, cell cycle progression and DNA synthesis were inhibited and caspase 3 activity and apoptosis were induced. Coinhibition of both pathways synergized to prevent cell cycle progression, inhibit DNA synthesis and induce apoptosis. These inhibitors had more apoptotic inducing effects when the cells were grown in response to IGF-1 than IL-3, indicating that IL-3 can induce additional anti-apoptotic pathways. These results demonstrate that the PI3K/Akt and Raf/MEK/ERK pathways are intimately involved in IGF-1R-mediated cell cycle progression and prevention of apoptosis in hematopoietic cells.     

Both src-dependent and -independent mechanisms mediate phosphatidylinositol 3-kinase regulation of colony-stimulating factor 1-activated mitogen-activated protein kinases in myeloid progenitors

Colony-stimulating factor 1 (CSF-1) supports the proliferation, survival, and differentiation of bone marrow-derived cells of the monocytic lineage. In the myeloid progenitor 32D cell line expressing CSF-1 receptor (CSF-1R), CSF-1 activation of the extracellular signal-regulated kinase (ERK) pathway is both Ras and phosphatidylinositol 3-kinase (PI3-kinase) dependent. PI3-kinase inhibition did not influence events leading to Ras activation. Using the activity of the PI3-kinase effector, Akt, as readout, studies with dominant-negative and oncogenic Ras failed to place PI3-kinase downstream of Ras. Thus, PI3-kinase appears to act in parallel to Ras. PI3-kinase inhibitors enhanced CSF-1-stimulated A-Raf and c-Raf-1 activities, and dominant-negative A-Raf but not dominant-negative c-Raf-1 reduced CSF-1-provoked ERK activation, suggesting that A-Raf mediates a part of the stimulatory signal from Ras to MEK/ERK, acting in parallel to PI3-kinase. Unexpectedly, a CSF-1R lacking the PI3-kinase binding site (DeltaKI) remained capable of activating MEK/ERK in a PI3-kinase-dependent manner. To determine if Src family kinases (SFKs) are involved, we demonstrated that CSF-1 activated Fyn and Lyn in cells expressing wild-type (WT) or DeltaKI receptors. Moreover, CSF-1-induced Akt activity in cells expressing DeltaKI is SFK dependent since Akt activation was prevented by pharmacological or genetic inhibition of SFK activity. The docking protein Gab2 may link SFK to PI3-kinase. CSF-1 induced Gab2 tyrosyl phosphorylation and association with PI3-kinase in cells expressing WT or DeltaKI receptors. However, only in DeltaKI cells are these events prevented by PP1. Thus in myeloid progenitors, CSF-1 can activate the PI3-kinase/Akt pathway by at least two mechanisms, one involving direct receptor binding and one involving SFKs.     

Activated Ha-ras induces apoptosis by association with phosphorylated Bcl-2 in a mitogen-activated protein kinase-independent manner.

Serum deprivation of Ha-ras-transformed brown adipocyte cell line resulted in a dramatic apoptotic cell death, as detected either by DNA laddering or by an increase in the percentage of hypodiploid cells or by nuclei condensation and fragmentation, as compared with immortalized cell line or primary fetal brown adipocytes. Moreover, transient transfection of immortalized brown adipocytes with a constitutively active ras gene (Ha-raslys12) mimics the high rate of apoptosis detected in the transformed cell line. On the other hand, transient transfection of the dominant-negative construct of raf-1 rescued serum-deprived Ha-ras-transformed brown adipocytes from apoptosis, decreasing the percentage of hypodiploid cells, the external display of phosphatidylserine, and the DNA laddering. However, inhibition of mitogen-activated protein kinase with PD098059 did not preclude apoptosis and in fact increased the rate of apoptosis observed in serum-deprived Ha-ras-transformed cells, indicating that the Ras/Raf-1 pathway induced apoptosis throughout a mitogen-activated protein kinase kinase 1 (MEK-1)-independent pathway. Furthermore, apoptosis in Ha-ras-transformed brown adipocytes is concurrent with an up-regulation in the expression of the pro-apoptotic protein Bcl-xS, the expression of the anti-apoptotic protein Bcl-2 being down-regulated. Finally, an association of Ras and Raf with phosphorylated Bcl-2 protein was demonstrated in immunoprecipitates from apoptotic cells. Thus, we propose a mechanism of apoptosis in Ha-ras-transformed adipocytes under serum deprivation involving Raf-1 association with phosphorylated Bcl-2, down-regulation of Bcl-2 expression, and up-regulation of Bcl-xS expression.     

Role of phosphoinositide 3-kinase in the aggressive tumor growth of HT1080 human fibrosarcoma cells

We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294-9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase-CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-kappaB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-kappaB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed in HT1080 cells, except for Raf and MEK, which were more active than HT1080 levels. This cross talk results in conversion to the aggressive tumorigenic phenotype. This latter observation is consistent with our previous observation that overstimulation of the activity of endogenous members of Ras signaling pathways, activated MEK in particular, is a prerequisite for aggressive tumorigenic growth.     

Modulation of intracellular signaling pathways to induce apoptosis in prostate cancer cells

An understanding of the molecular pathways defining the susceptibility of prostate cancer, especially refractory prostate cancer, to apoptosis is the key for developing a cure for this disease. We previously demonstrated that up-regulating Ras signaling, together with suppression of protein kinase C (PKC), induces apoptosis. Dysregulation of various intracellular signaling pathways, including those governed by Ras, is the important element in the development of prostate cancer. In this study, we tested whether it is possible to modulate the activities of these pathways and induce an apoptotic crash among them in prostate cancer cells. Our data showed that DU145 cells express a high amount of JNK1 that is phosphorylated after endogenous PKC is suppressed, which initiates caspase 8 cleavage and cytochrome c release, leading to apoptosis. PC3 and LNCaP cells contain an activated Akt. The inhibition of PKC further augments Akt activity, which in turn induces ROS production and the accumulation of unfolded proteins in the endoplasmic reticulum, resulting in cell death. However, the concurrent activation of JNK1 and Akt, under the condition of PKC abrogation, dramatically augment the magnitude of apoptosis in the cells. Thus, our study suggests that Akt, JNK1, and PKC act in concert to signal the intracellular apoptotic machinery for a full execution of apoptosis in prostate cancer cells.